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The Distribution of Cytochrome C in Developing Pollen of Normal and S Male-sterile Maize

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Title:
The Distribution of Cytochrome C in Developing Pollen of Normal and S Male-sterile Maize
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Verduin, Lindsey
Chase, Christine ( Mentor )
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Gainesville, Fla.
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University of Florida
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English

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The Distribution of Cytochrome C in Developing Pollen of Normal and S
Male-sterite Maize

Lindsey Verduin and Christine D. Chase


ABSTRACT


Apoptosis, or programmed cell death, is a known regulatory feature of eukaryotic cells. In mammalian

cells, apoptosis is often initiated by the release of cytochrome c from the mitochondria. However, it is

unknown whether cytochrome c plays a role in cell death signaling in plants. We hypothesized cytochrome c

released from the mitochondria causes pollen collapse in S male-sterile maize. Mitochondrial pellet and

post-mitochondrial supernatant samples were collected from developing sterile and normal pollen. The location

and relative abundance of cytochrome c in these samples was detected by Western blot analysis. A

large concentration of cytosolic cytochrome c was detected in both sterile and normal samples, contradictory to

the expected lack of the protein in normal cytosolic fractions. This finding indicates that cytochrome c release

from mitochondria does not, by itself, initiate cell death.



INTRODUCTION


Apoptosis, or programmed cell death, is a mechanism found in vertebrates allowing individual cells to die

and disintegrate in a regulated process. While apoptosis is often associated with disease, it is also a

necessary cellular function, involved in the immune response and the destruction of worn out cells1. Cell death can

be induced by a number of factors but the main executers in all apoptotic pathways are caspases. Caspases are

a family of proteases that, once activated, initiate a caspase cascade, acting on various cellular substrates,

to culminate in cell death2.


Various signals and organelles can induce apoptosis, but perhaps the most well known cell death pathway

involves the mitochondria. Proapoptotic proteins, such as Bax and Bid of the Bcl-2 family, interact with the

outer mitochondrial membrane, forming openings in the mitochondria3. This leads to mitochondrial

depolarization, swelling, and ultimately, the release of its proteins3. The most significant protein released

is cytochrome c, which is part of the oxidative phosphorylation pathway. Upon its release, cytochrome binds

with Apaf-1, forming an apoptosome2. This complex activates caspase 9, initiating the "death cascade"2.


While apoptosis is a common feature in mammalian cells, it is unknown whether plants, specifically maize, follow




the same apoptotic cell death pathway. As in animals, plant cell death is a common regulatory process, important

in a plant's sexual maturation and the growth of numerous structures4. Insight into this issue could be found

with studies on maize cytoplasmic male sterility (CMS). Cytoplasmic male sterility is an occurrence in many

plants that results in the production of nonfunctional pollen5. The cause of pollen collapse has not yet

been determined. However, the expression of two mitochondrial reading frames unique to CMS-S maize, orf355

and orf77, may be responsible for pollen collapse in this system5. The exact mechanism of orf355 and orf77

sterility is unknown. Transcription and translation of orf77 can result in a truncated protein called orfl75. Orfl7

has homology with the mitochondrial ATP synthase protein ATP9, specifically in the transmembrane domain. It

is thought that orf355 and orf77 may disrupt the assembly of ATP synthase leading to pore formation in

the mitochondrial membrane and the subsequent release of mitochondrial proteins5.



The purpose of this research was to determine the location and relative abundance of cytochrome c in CMS-S

maize cells. From this, it could be possible to ascertain whether cytochrome c plays a role in cell death. A

study conducted by Lee et al. pertaining to pollen abortion in CMS maize described mitochondrial swelling prior

to pollen cell death, which is similar in morphology to mitochondria in mammalian cells undergoing apoptosis6.

Based on this morphological similarity and Western blot results showing collapsed pollen mitochondria depleted

of cytochrome c, we hypothesized that cytochrome c could be a cell death signal in plants. Our hypothesis

predicts that if cytochrome c acts a cell death signal, the majority of cytochrome c should be located in the cytosol

of collapsed pollen, while normal pollen granules should have cytochrome c concentrated within the mitochondria.



METHODS


Cell fractionation


Fig. 1 shows the procedure for a cell fractionation. Zea mays (corn) pollen was obtained from Mo17N and

Mo17S lines, normal and sterile respectively. Pollen was recovered from each line at various stages of the corn's

life cycle, including normal starch filling pollen, sterile collapsed pollen, and normal and sterile microspores.

The microspore is an early stage in pollen development. Mo17S and Mo17N microspores are

morphologically indistinguishable. Mo17S pollen collapses abruptly later in pollen development.



A pollen sample (1-2g) was crushed in mitochondrial grinding buffer (0.5M sucrose, 50mM Trizma base, imM

EGTA, 10mM KH2PO4, pH 7.6; BSA, 2-mercaptoethanol, 0.1M PMSF) using a mortar and pestle. Following

the protocol, two filtrations were performed to isolate cellular components. The filtrate was centrifuged three

times, with the centrifugations being performed on each successive supernatant collected. The end product was

a mitochondrial pellet sample and a post-mitochondrial supernatant sample. 10% of each mitochondrial pellet

and supernatant fraction was examined to determine relative cytochrome c abundance among the samples.







300 ul crude exht
+ 75 l 6X NUPAGE $8
70oC 10 min
15,00 x g, nrm tamp, 10 min
Recove sup for g hez:
38 ul = 1% staring marnal
Fiscal pew

300 I 500 g sup
+ 5 u SX NUPAGE SB

Recover sup for gel; free;
38 Lu= 1% Wtarting meal



Discard npet
+ 75 A 5X NUPAGE SB
70cC 10 fmin
16 000 N g. room amp 10 min
Reower sup for gel; freeze;
38 ul= 1% starting male
Discapollet


3000 ul aude etrat



2,700 ul crude extact
(2 x 1,350 li)
1500 x g, 4oC 15min



2,400 ul 1,5 g sup
(2 x 1.200 ul)
15,001 xg.4oC, 15 min




2I li lIOO g sup
= post todr~ su
00 NOT WI ZE
Prep for BNGEe
!TinnOpeip#tein


1,0sg( pellets
+ 150 X NUPAGE SB eat
70oC 10 fin
16.000 xg room temp, 10 min
Rmxw sup for gl: freeze 3.1 ul =1% starig medal



IS 00 g Wcletfs = nitodthrdriar
+ 150 ul iI NUPAGE 58 e3th
70oC 10 min
16,000 x g room ep, 10 min
Rewr sup 'of gel ftrze 3 8 I a 1%
Starbng material
iscard pelet


Figure 1. Cell fractionation diagram



Gel electrophoresis


The protein samples obtained from cell fractionations were separated on 12% NuPAGE� Bis-Tris gel

(Invitrogen; Carlsbad, CA). Varying amounts of protein samples (3.8 - 25 ul) were mixed with lx sample buffer

(5x sample buffer, DTT, 0.1% bromphenol blue, 0.1% PMSF) and loaded on the gel. Molecular weight

markers (SeeBlue5 Plus2 Pre-Stained Standard) were loaded in lane 1 of every gel. The gel was run in lx

NuPAGE� MES SDS Running Buffer, with NuPAGE� antioxidant added to the inner chamber, according

to manufacturer's instructions (XCell Sure LockTM Mini-Cell). Electrophoresis was carried out at 100V for about

2 hours, or until dye marker reached 2 cm from the bottom of the case.



Western blotting


Proteins from the gel were transferred to a nitrocellulose membrane (Invitrogen 0.2 pm pore nitrocellulose/

filter paper sandwich; XCell IITM Blot Module). The blot was incubated in blocking solution (2% or 3% w/v BSA

in TBST) for 1 hour, washed in TBST (NaCI 0.14M; KCI 2.7mM; 1M Tris, pH8), and incubated 1 hour in

primary antibody. Cytochrome c monoclonal primary antibody was obtained from PharMingen (clone

7H8.2C12). ATPy was an affinity purified polyclonal primary antibody. Cytochrome c and ATPy were both diluted

at 10pl/50ml TBST. The blot was washed 6 times in TBST in 10 min intervals, followed by incubation for 1 hour in

a horseradish peroxidase linked secondary antibody (mouse or rabbit at lpl/40ml TBST). The blot was again

washed 6 times in TBST. Reactivity was observed using chemiluminescence (SuperSignal, Pierce)

and autoradiographic film.




RESULTS



Figure 2.1 shows an anti-cytochrome c blot. Comparison with the molecular weight markers showed a 14





kDa protein, which was in keeping with the known weight of cytochrome c (12 kDa). The relative abundance
of cytochrome c in each lane was compared between sterile and normal samples at distinct life stages. Sterile
and normal immature ear samples were included to determine if cytochrome c release from the mitochondria
could be a developmental feature in the maize life cycle. The sterile and normal immature ear and
microspore samples showed no difference in cytochrome c abundance in either the pellet or the supernatant.
An unexpected result was the observation of large amounts of cytosolic cytochrome c in all the samples.
However, sterile collapsed pollen does appear to have the highest abundance of cytochrome c in the cytosol.


Figure 2.2 shows an anti- ATPy blot. ATPy is a subunit of ATP synthase, which is a mitochondrial inner
membrane structure. The ATPy blot was performed on the blot from Fig. 2.1 and included as a control to
assess membrane integrity. The predominance of ATPy in the pellet samples shows that the mitochondria are
largely intact. The BSA present in supernatant fractions caused artifactual lane narrowing for the
supernatant fractions.





2 3 4 5 6 7 8 9 10 11



14 kDa





32 kDa





Figure 2.1. Results of Western blot against cytochrome c. Lane 2,3: sterile immature ear
mitochondrial pellet (P) and post-mitochondrial supernatant (S). Lane 4,5: normal immature ear P and
S. Lane 6,7: sterile microspore (msp) P and S. Lane 8,9: normal msp P and S. Lane 10,11:
sterile collapsed pollen P and S. All samples were prepared in 0.5M sucrose and 1% protease-free BSA.


Figure 2.2. Results of Western blot against ATPy. Lane samples correspond to Fig. 2.1.



Figure 3 shows results from an anti-cytochrome c blot. The samples in Fig. 3 were prepared in delipidated BSA,
as opposed to 1% protease-free BSA. Delipidated BSA is known to protect membrane integrity, prompting us
to compare with protease-free BSA to determine if the delipidated BSA affected cytochrome c release. The
results from Fig. 3 are identical to Fig. 2.1, indicating that the source of BSA has no effect on cytochrome c
release. Fig. 3 shows more clearly the unexpectedly high amount of cytosolic cytochrome c in the normal
microspore and starch filling pollen. However, there is a striking difference evident between sterile collapsed





pollen and normal starch filling pollen cytochrome c abundance. The majority of cytochrome c in collapsed pollen
is released into the cytosol, while starch filling pollen has an equal proportion of cytochrome c in its mitochondria
and cytosol.


According to a study by Petrussa et al., when mitochondria are suspended in a medium containing potassium,
the mitochondrial K+ATP channel is opened, causing the organelle to swell and release cytochrome c7. Based on
this fact, it was thought that potassium from the KH2PO4 found in the mitochondrial grinding buffer might have
been affecting the amount of cytochrome c released from the mitochondria. Lanes 11 and 12 of Figure 3 show
sterile microspore samples prepared in grinding buffer containing no KH2PO4. The absence of potassium had no
effect on cytochrome c abundance, as can be seen by comparing lanes 11 and 12 with lanes 2 and 3.




2 3 4 5 6 7 8 9 10 1112


14 kDa





Figure 3. Results of Western blot against cytochrome c. Lanes 2,3: sterile msp P and S. Lanes 4,5:
normal msp P and S. Lanes 7,8: sterile collapsed pollen P and S. Lanes 9,10: normal starch filling pollen
P and S. Lanes 11,12: sterile msp P and S prepared in potassium free grinding buffer. All samples
were prepared in 0.5M sucrose and delipidated BSA.



The abundance of cytochrome c in the supernatant of the normal starch filling pollen was unexpected. The
sucrose concentration in the mitochondrial grinding buffer was altered to assess whether it played any role
in cytochrome c release. Figure 4 shows an anti-cytochrome c blot on sterile and normal microspores prepared
in 0.065M and 0.8M sucrose concentration. Increasing the sucrose concentration from 0.5M did not have any
effect on the amount of cytochrome c released in the samples.




2 3 4 5 6 7 8 9 10 11


14 kDa





Figure 4. Results of Western blot against cytochrome c. Lanes 2,4: sterile msp P and S in 0.65M
sucrose. Lanes 6,8: normal msp P and S in 0.65M sucrose. Lanes 10,11: normal msp P and S in




0.8M sucrose. All samples were prepared using delipidated BSA.


DISCUSSION


The hypothesis that cytochrome c is a plant cell death signal was not fully demonstrated. While sterile

collapsed pollen mitochondria were found to be largely void of cytochrome c, this does not conclusively indicate

that cytochrome c is causing the pollen collapse. The finding that both collapsed pollen and normal starch

filling pollen have large amounts of cytochrome c in the cytosol indicate that cytochrome c may not play a role in

cell death signaling in plants. This theory is supported in a recent study by Yao et al. which showed that

while cytochrome c is released during plant cell death, cytochrome c itself is not sufficient to induce apoptosis8.



The finding that sterile and normal microspores showed the same cytochrome c abundance pattern (a large

amount present in the cytosol) was unexpected. As mentioned above, the presence of a large cytosolic cytochrome

c concentration in normal starch filling pollen was surprising. These results could have been caused by various

factors not taken into previous consideration. The three experiments performed to account for these

discrepancies, removing potassium from the grinding buffer, increasing sucrose concentrations, and substituting

1% protease BSA, had no effect on cytochrome c concentrations.



One aspect that may be relevant to future experimentation is the form of cytochrome c that is present in the

cytosolic samples. Newly synthesized cytochrome c present in the cytoplasm is in the "apo" form. The "apo"

form contains no heme group and is not functional until it is transported into the mitochondria. Once inside

the mitochondria, a heme group is added and cytochrome c is converted to the "holo" form9.



The hypothesis predicts that the cytochrome c in microspore supernatants would be in the "apo" form,

while collapsed pollen supernatant should be mainly "holo" cytochrome c. A series of experiments utilizing the

ability of heme to direct chemiluminescence was conducted. The above mentioned protocol of fractionation

and electroblotting was followed to produce new cellular samples. SuperSignal (Pierce) luminol was added directly

to the nitrocellulose membrane in the absence of antibodies10. However, the chemiluminescent reaction was

not sensitive enough to react under our extraction conditions. This topic of study will pave the way for a new line

of experimentation that is capable of detecting and distinguishing the "apo" and "holo" form of cytochrome c.

Future experiments will be performed involving laser confocal microscopy to pinpoint where exactly the cytochrome

c is located within the cell9.



Discovering the intricate pathways of plant cell death has important agricultural and economic implications.

By understanding how plant death occurs, it may be possible to prevent or induce cell death, depending upon

the situation. Knowledge concerning plant cell death is a gray area but full of numerous avenues of study to

unravel its causes and pathways.







REFERENCES


1. Lemasters JJ. Dying a thousand deaths: redundant pathways from different organelles to apoptosis and

necrosis. Gastroenterology 2005;129:351-60.

2. Lavrik IN, Golks A, Krammer PH. Caspases: pharmacological manipulation of cell death. Journal of Clinical

Investment 2005;115:2665-72.

3. Green GR, Kroemer G. The pathophysiology of mitochondrial cell death. Science 2004;305:626-29.

4. van Doorn WG, Woltering EJ. Many ways to exit? Cell death categories in plants. Trends in Plant

Science 2005;10:117-22.

5. Gallagher LJ, Betz SK, Chase CD. Mitochondrial RNA editing truncates a chimeric open reading frame associated

with S male-sterility in maize. Current Genetics 2002;42:179-84.

6. Lee SJ, Earle ED, Gracen VE. The cytology of pollen abortion in S cytoplasmic male-sterile corn anthers.

American Journal of Botany 1980;67:237-45.

7. Petrussa E, Casolo V, Peresson C, Braidot E, Vianello A, Marci F. The K+ATP channel is involved in a low-

amplitude permeability transition in plant mitochondria. Mitochondrion 2004;3:297-307.

8. Yao N, Eisfelder BJ, Marvin J, Greenberg JT. The mitochondrion- an organelle commonly involved in programmed

cell death in IArabidopsis thaliana. The Plant Journal 2004;4:596-610.

9. Oliver L, LeCabellec MT, Pradal G, Meflah K, Kroemer G, Vallette FM. Constitutive presence of cytochrome c in

the cytosol of a chemoresistant leukemic cell line. Apoptosis 2005;10:277-87.

10. Vargas C, McEwan AG, Downie JA. Detection of c-type cytochromes using enhanced chemiluminescence.

Analytical Biochemistry 1993;209:219-23.





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